Mitochondria are dynamic membrane organelles that undergo division and fusion in highly regulated manners. The balance between these opposing activities plays a critical role in controlling mitochondrial structure and function. Mitochondrial division and fusion are mediated by conserved dynamin-related GTPases that include dynamin-related protein 1 (Drp1) for division, and mitofusin and optic atrophy 1 (Opa1) for fusion. Inhibition of mitochondrial division increases the size of the mitochondria due to ongoing fusion, whereas inhibition of fusion leads to fragmentation of the mitochondria. Abnormalities in mitochondrial division and fusion are associated with many neurodegenerative diseases, such as autosomal dominant optic atrophy, Charcot-Marie-Tooth neuropathy, Alzheimer's disease, Huntington's disease, and Parkinson's disease. Understanding the pathogenesis of these diseases requires a deeper knowledge of the molecular mechanism of mitochondrial dynamics. In this proposed research, we will identify and characterize novel proteins that bind to and regulate the central mitochondrial division protein Drp1. We have been developing innovative approaches to achieve this goal by combining two technologies - in vitro protein-protein interaction analysis using functional protein microarrays and in vivo protein-protein interaction analysis using the chemically inducible hetero-dimerization system consisting two proteins, the FK506-binding protein (FKBP) and the rapamycin-binding domain of mTOR (FRB). Using protein microarrays, we have performed a genome- wide search and identified 18 Drp1-binding proteins. In this revision, we will validate their interactions with Drp1 in live cells using the FBP-FRB hetero-dimerization system. Finally, we will determine their functional importance in mitochondrial division using gene knockdown approaches. Therefore, this study will provide a novel mechanistic insight into mitochondrial division.